Todd G. Cook

IFATS Collection: Human Adipose Tissue‐Derived Stem Cells Induce Angiogenesis and Nerve Sprouting Following Myocardial Infarction, in Conjunction with Potent Preservation of Cardiac Function

The administration of therapeutic cell types, such as stem and progenitor cells, has gained much interest for the limitation or repair of tissue damage caused by a variety of insults. However, it is still uncertain whether the morphological and functional benefits are mediated predominantly via cell differentiation or paracrine mechanisms. Here, we assessed the extent and mechanisms of adipose‐derived stromal/stem cells (ASC)‐dependent tissue repair in the context of acute myocardial infarction. Human ASCs in saline or saline alone was injected into the peri‐infarct region in athymic rats following left anterior descending (LAD) coronary artery ligation. Cardiac function and structure were evaluated by serial echocardiography and histology. ASC‐treated rats consistently exhibited better cardiac function, by all measures, than control rats 1 month following LAD occlusion. Left ventricular (LV) ejection fraction and fractional shortening were improved in the ASC group, whereas LV remodeling and dilation were limited in the ASC group compared with the saline control group. Anterior wall thinning was also attenuated by ASC treatment, and post‐mortem histological analysis demonstrated reduced fibrosis in ASC‐treated hearts, as well as increased peri‐infarct density of both arterioles and nerve sprouts. Human ASCs were persistent at 1 month in the peri‐infarct region, but they were not observed to exhibit significant cardiomyocyte differentiation. Human ASCs preserve heart function and augment local angiogenesis and cardiac nerve sprouting following myocardial infarction predominantly by the provision of beneficial trophic factors. STEM CELLS 2009;27:230–237

Suppression of Hepatocyte Growth Factor Production Impairs the Ability of Adipose‐Derived Stem Cells to Promote Ischemic Tissue Revascularization

The use of adipose‐derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual‐cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC‐shHGF and ASC‐shCtrl, respectively). Transduced ASC‐shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC‐shHGF were also significantly impaired, compared with ASC‐shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP+ cells were detected at 3 weeks in ischemic limbs of mice treated with ASC‐shHGF compared with those treated with ASC‐shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell‐based therapies.

17β-Estradiol mediates superior adaptation of right ventricular function to acute strenuous exercise in female rats with severe pulmonary hypertension.

17β-Estradiol (E2) exerts protective effects on right ventricular (RV) function in pulmonary arterial hypertension (PAH). Since acute exercise-induced increases in afterload may lead to RV dysfunction in PAH, we sought to determine whether E2 allows for superior RV adaptation after an acute exercise challenge. We studied echocardiographic, hemodynamic, structural, and biochemical markers of RV function in male and female rats with sugen/hypoxia (SuHx)-induced pulmonary hypertension, as well as in ovariectomized (OVX) SuHx females, with or without concomitant E2 repletion (75 μg·kg(-1)·day(-1)) immediately after 45 min of treadmill running at 75% of individually determined maximal aerobic capacity (75% aerobic capacity reserve). Compared with males, intact female rats exhibited higher stroke volume and cardiac indexes, a strong trend for better RV compliance, and less pronounced increases in indexed total pulmonary resistance. OVX abrogated favorable RV adaptations, whereas E2 repletion after OVX markedly improved RV function. E2s effects on pulmonary vascular remodeling were complex and less robust than its RV effects. Postexercise hemodynamics in females with endogenous or exogenous E2 were similar to hemodynamics in nonexercised controls, whereas OVX rats exhibited more severely altered postexercise hemodynamics. E2 mediated inhibitory effects on RV fibrosis and attenuated increases in RV collagen I/III ratio. Proapoptotic signaling, endothelial nitric oxide synthase phosphorylation, and autophagic flux markers were affected by E2 depletion and/or repletion. Markers of impaired autophagic flux correlated with endpoints of RV structure and function. Endogenous and exogenous E2 exerts protective effects on RV function measured immediately after an acute exercise challenge. Harnessing E2s mechanisms may lead to novel RV-directed therapies.

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6

Objective: Bone marrow‐derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose‐derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA‐transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony‐forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS‐induced myelosuppression. This effect was specifically dependent on the anti‐inflammatory factor TSG‐6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG‐6 may be novel approaches to reverse CS‐induced myelosuppression. Stem Cells 2015;33:468–478

Human Adipose-Derived Stem Cells Suppress Elastase-Induced Murine Abdominal Aortic Inflammation and Aneurysm Expansion Through Paracrine Factors.

Abdominal aortic aneurysm (AAA) is a potentially lethal disease associated with immune activation-induced aortic degradation. We hypothesized that xenotransplantation of human adipose-derived stem cells (hADSCs) would reduce aortic inflammation and attenuate expansion in a murine AAA model. Modulatory effects of ADSCs on immune cell subtypes associated with AAA progression were investigated using human peripheral blood mononuclear cells (hPBMNCs) cocultured with ADSCs. Murine AAA was induced through elastase application to the abdominal aorta in C57BL/6 mice. ADSCs were administered intravenously, and aortic changes were determined by ultrasonography and videomicrometry. Circulating monocytes, aortic neutrophils, CD28− T cells, FoxP3+ regulatory T cells (Tregs), and CD206+ M2 macrophages were assessed at multiple terminal time points. In vitro, ADSCs induced M2 macrophage and Treg phenotypes while inhibiting neutrophil transmigration and lymphocyte activation without cellular contact. Intravenous ADSC delivery reduced aneurysmal expansion starting from day 4 [from baseline: 54.8% (saline) vs. 16.9% (ADSCs), n = 10 at baseline, n = 4 at day 4, p < 0.001], and the therapeutic effect persists through day 14 (from baseline: 64.1% saline vs. 24.6% ADSCs, n = 4, p < 0.01). ADSC administration increased aortic Tregs by 20-fold (n = 5, p < 0.01), while decreasing CD4+CD28− (-28%), CD8+CD28− T cells (-61%), and Ly6G/C+ neutrophils (-43%, n = 5, p < 0.05). Circulating CD115+CXCR1−LY6C+-activated monocytes decreased in the ADSC-treated group by day 7 (-60%, n = 10, p < 0.05), paralleled by an increase in aortic CD206+ M2 macrophages by 2.4-fold (n = 5, p < 0.05). Intravenously injected ADSCs transiently engrafted in the lung on day 1 without aortic engraftment at any time point. In conclusion, ADSCs exhibit pleiotropic immunomodulatory effects in vitro as well as in vivo during the development of AAA. The temporal evolution of these effects systemically as well as in aortic tissue suggests that ADSCs induce a sequence of anti-inflammatory cellular events mediated by paracrine factors, which leads to amelioration of AAA progression.

Pulmonary Retention of Adipose Stromal Cells following Intravenous Delivery is Markedly Altered in the Presence of ARDS

Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (~7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (~3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS‐ASC) and smokers (CS‐ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS‐ASC improved perfusion in ischemic limbs, CS‐ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS‐ASC and ASC from CS–exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS‐ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS‐ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell‐derived growth factor 1 (SDF‐1). Conversely, CS‐ASC secreted the angiostatic/pro‐inflammatory factor Activin A, which was not detected in nCS‐ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS‐ASC cocultures than in EC/nCS‐ASC cocultures. CS‐ASC also responded to inflammatory cytokines with 5.2‐fold increase in Activin A secretion, whereas nCS‐ASC showed minimal Activin A induction. Supplementation of EC/CS‐ASC cocultures with nCS‐ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF‐1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS‐ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856–867

Human Adipose-Derived Stem Cells Ameliorate Cigarette Smoke-Induced Murine Myelosuppression via Secretion of TSG-6: Adipose Stem Cells Ameliorate Smoking-Induced Myelosuppression Via TSG-6

Objective: Bone marrow‐derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose‐derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA‐transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony‐forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS‐induced myelosuppression. This effect was specifically dependent on the anti‐inflammatory factor TSG‐6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG‐6 may be novel approaches to reverse CS‐induced myelosuppression. Stem Cells 2015;33:468–478

Human Adipose-derived Stem Cells Ameliorate Cigarette Smoke-induced Murine Myelosuppression via TSG-6

Objective: Bone marrow‐derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose‐derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA‐transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony‐forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS‐induced myelosuppression. This effect was specifically dependent on the anti‐inflammatory factor TSG‐6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG‐6 may be novel approaches to reverse CS‐induced myelosuppression. Stem Cells 2015;33:468–478